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bioRxiv · 10.1101/2019.12.19.882050

Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq

Qiu, Q.·Hu, P.·Govek, K. W.·Camara, P. G.·Wu, H.
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Abstract

Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal dynamics of RNA biogenesis and decay. Here we present single-cell new transcript tagging sequencing (scNT-Seq), a method for massively parallel analysis of newly-transcribed and pre-existing RNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking metabolically labeled newly-transcribed RNAs with T-to-C substitutions. By simultaneously measuring new and old transcriptomes, scNT-Seq reveals neuronal subtype-specific gene regulatory networks and time-resolved RNA trajectories in response to brief (minutes) versus sustained (hours) neuronal activation. Integrating scNT-Seq with genetic perturbation reveals that DNA methylcytosine dioxygenases may inhibit stepwise transition from pluripotent embryonic stem cell state to intermediate and totipotent two-cell-embryo-like (2C-like) states by promoting global RNA biogenesis. Furthermore, pulse-chase scNT-Seq enables transcriptome-wide measurements of RNA stability in rare 2C-like cells. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.

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Qiu, Q., Hu, P., Govek, K. W., Camara, P. G., Wu, H.. 2019-12-20. Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq. https://doi.org/10.1101/2019.12.19.882050

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